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Introduction

Procedure Overview

The following protocols from Molecular Research Center, Inc., (MRC) describe sequential isolation of DNA and protein from the interphase and organic/phenol phase of the lysate from the TRI Reagent® * RNA Isolation Protocol.

The RNA Isolation Protocol is provided with TRI Reagent (Cat #9738) and is available at: http://www.ambion.com

The DNA isolated by this protocol is suitable for PCR, restriction enzyme digestion, and Southern blotting. The recovered protein is suitable for analysis by Western blotting.

Protocol - TRI Reagent DNA Isolation

DNA is precipitated from the phenol phase and interphase of samples that have been homogenized (or lysed) in 1 ml of TRI Reagent (step 5 in the RNA Isolation Protocol). After a series of washes to remove residual phenol, the DNA pellet is solubilized in a mild alkaline solution, and the pH is adjusted. This technique performs well with samples containing >10 μg of DNA.

The molecular weight of the recovered DNA depends on the shearing forces applied during homogenization. If recovery of high molecular weight DNA is desired, use a loosely fitting homogenizer in the initial homogenization step of the RNA Isolation Protocol. Avoid using a Polytron homogenizer.

If the DNA is isolated only for quantitative purposes: a) samples can be more vigorously homogenized, including the use of a Polytron; b) the phenol phase and interphase can be stored at 4°C for a few days or at –70°C for a few months; c) the DNA can be solubilized using 40 mM NaOH instead of an 8 mM solution, and by vortexing the DNA pellet instead of pipetting.

Special Handling Precautions

The starting material for this procedure contains TRI Reagent, which contains a poison (phenol) and an irritant (guanidine thiocyanate). Contact with TRI Reagent will cause burns and can be fatal. Use gloves and other personal protection when working with TRI Reagent.

DNA Precipitation

This procedure begins with the material remaining after step 5 of the TRI Reagent RNA Isolation Protocol, i.e., the lower, red, organic/phenol phase, and the interphase. The phenol phase and interphase can be stored at 4°C overnight before proceeding with the DNA isolation procedure.

Remove any aqueous phase remaining over the interphase

Remove any remaining aqueous phase overlying the interphase

NOTE Careful removal of any residual aqueous phase is critical for the quality of the isolated DNA.

If your sample contains <10 μg DNA, see the alternate protocol

Add 300 μl of 100% ethanol and mix by inversion

Add 300 μl of 100% ethanol per 1 ml of TRI Reagent used for the initial homogenization.

Mix samples by inversion.

Incubate at room temp for 2–3 min, centrifuge at 2,000 x g for 5 min at 4°C, and remove the supernatant

Assess DNA Yield and Quality

Determine the DNA concentration and purity by diluting an aliquot of the preparation in water or buffer with a pH>7.5 and reading the absorbance in a traditional spectrophotometer at 260 nm and at 280 nm.

To determine the DNA concentration in μg/ml, multiply the A 260 by the dilution factor and the extinction coefficient
(1 A 260 = 50 μg double-stranded DNA/ml).

A260 X dilution factor X 50 = μg DNA/ml

DNA yield and size can vary considerably between samples. Yield is dependent on factors such as sample type, health of the organism, and thoroughness of sample disruption. Sample handling has a strong impact on the size of the recovered DNA. The DNA yield and size expectations listed below assume ideal conditions for factors that the user can control.

Add back extraction buffer to the interphase-organic phase mixture. Use 500 μl of back extraction buffer per 1 ml of TRI Reagent used for the initial homogenization.

Vigorously mix the sample by inversion for 15 sec and incubate for 10 min at room temperature.

Centrifuge at 12,000 x g for 15 min at 4°C to separate the phases.

Transfer the upper, aqueous phase containing DNA to a clean tube and save the interphase and organic phase at 4°C for subsequent protein isolation.

(Optional) If the expected DNA yield is less than 20 μg, add 2–8 μl of acrylamide carrier to the aqueous phase and mix.

Add 400 μl of isopropanol per 1.0 ml of TRI Reagent used for the initial homogenization to precipitate DNA from the aqueous phase. Mix the sample by inversion and incubate for 5 min at room temperature.

Sediment the DNA by centrifugation at 12,000 x g for 5 min at 4–25°C and remove the supernatant.

TRI Reagent Protein Isolation Protocol

Protein is precipitated from the phenol-ethanol supernatant obtained after sedimentation of the DNA pellet. After a series of washes, the protein-containing pellet is solubilized in a suitable detergent-containing solvent.

The procedure is carried out at room temperature unless stated otherwise.

Special Handling Precautions

The starting material for this procedure contains TRI Reagent, which contains a poison (phenol) and an irritant (guanidine thiocyanate). Contact with TRI Reagent will cause burns and can be fatal. Use gloves and other personal protection when working with TRI Reagent.

After dispersing the pellet, add another 500 μl of Protein Wash 1 to the sample.

Incubate for 10 min at room temperature.

Centrifuge the sample at 8,000 x g for 5 min, anddiscard the supernatant

Sediment the protein by centrifugation at 8,000 x g for 5 min.

Remove and discard the supernatant.

Wash two more times with 1 ml Protein Wash 1

These washes remove residual phenol.

Add 1 ml Protein Wash 1 to the pellet and disperse the pellet by vortexing.

Incubate for 10 min at room tempterature.

Sediment the protein by centrifugation at 8,000 x g for 5 min, and remove and discard the supernatant.

Repeat steps a–c to wash a second time with 1 ml Protein Wash 1.

STOPPING POINT - In general, protein pellets suspended in Protein Wash 1 or in Protein Wash 2 can be stored for at least one month at 4°C or one year at –20°C. Individual proteins may display different sensitivity to long-term storage; establish optimal storage conditions for sensitive and labile proteins.

Wash with 1 ml Protein Wash 2 for 10 min

Disperse the pellet in 1 ml Protein Wash 2.

Incubate for 10 min.

Centrifuge the sample at 8,000 x g for 5 min at 4°C.

Remove and discard the supernatant, invert the tube and dry the pellet for 7–10 min at room temperature.

Protein Solubilization

Add 200 μl of solvent per 10–20 mg of tissue sample to the protein pellet. Use a solvent such as 1% SDS, 10 M urea, or another suitable detergent-based solvent. The solubility and stability of specific proteins can be influenced by different detergent solutions. Determine which works best for your experimental needs by solubilizing small samples in different solvents. Addition of a reducing agent such as tributylphosphine (2.5% of solution volume) will improve protein yield in most preparations.

Gently disperse and solubilize the pellet for 15–20 min by “flicking” the tube or pipetting.

STOPPING POINT
Samples may be stored at –20˚C at this point.

Prior to use, heat the protein sample for 3 min at 100°C, centrifuge at 10,000 x g for 5 min, and transfer the supernatant to a clean tube

If the sample has been stored at –20°C, thaw at 25°C for 10–15 min before proceeding. Solubilized protein may form insoluble aggregates during storage.

Prior to use in Western analysis, heat the protein sample for 3 min at 100°C.

Centrifuge the sample at 10,000 x g for 5 min at room temperature to sediment any insoluble material.